Integrative Extraction of Ergosterol, （1→3）-α-D-Glucan and Chitosan from Penicillium chrysogenum Mycelia

Abstract Ergosterol,（1→3）-α-D-glucan and chitosan are important biomaterials. In this research, a process has been developed to integratively extract ergosterol, （1→3）-α-D-glucan, and chitosan from Penicillium chrysongenum mycelium. First the mycelia are pretreated with 0.1mol·L^-1 of NaOH. After recovery by centrifugation the solid portion is made to undergo saponification and deacetylation reactaons by addition of 2mol·L^-1 NaOH and et anol.After reaction, extraction is carried out by addition of petroleum ether, which separates the reaction mixture into two phases. The upper layer of petroleum ether contains extracted ergosterol, and the .bottom layer of NaOH solution contains （1→3）-α-DEglucan; the chitosan is on the mycelia residuum. After isolation, the recovery yield of ergosterol is 0.52% of dry mycelium. That of （1→3）-α-D-glucan is about 8.2%; and chitosan is 5.7% with 86% deacetylation. The compositions have been characterized by 1R, HPLC analyses.

SOLUTION PROPERTIES OF ANTITUMOR CARBOXYMETHYLATED DERIVATIVES OF α-(1→3)-D-GLUCAN FROM GANODERMA LUCIDUM

Fractions of a water insoluble α-(1→3)-D-glucan (GL) extracted from Ganoderma lucidum were carboxy-methylated (CM) to obtain water-soluble carboxymethylated derivatives (CM-GL) having a degree of substitution (DS) of0.38～0.51. Weight-average molecular weight M_w and intrinsic viscosity [η] of the samples CM-GL were measured by gelpermeation chromatography combined with laser light scattering (GPC-LLS) and viscometry. The CM-GL exhibits a stifferchain in aqueous solution at 25℃ than the original glucan, The antitumor activities against Ehrlich ascites carcinoma (EAC,5×10~6) of the carboxymethylated derivatives from the α-glucan and curdlan, a β-glucan, are significantly higher than thoseof the original glucans. The effects of the relatively low molecular weight, expanded chains and better water-solubility of theCM-GL on the enhancement of antitumor activity could not be neglected. The chain stiffness decreased speedily withincrease of temperature from 40 to 60℃ or NaOH concentration from 0.1 to 0.4 in the solution, respectively, and the changeof the chain stiffness is reversible.

STRUCTURE,MOLECULAR WEIGHT AND BIOACTIVITIES OF(1→3)-β-D-GLUCANS AND ITS SULFATED DERIVATIVES FROM FOUR KINDS OF LENTINUS EDODES

Lentinan samples,(1→3)-β-D-glucans containing 4.6-15.2 wt% proteins,coded as L-I_1 L-I_2 L-I_3 and L-I_4(L-I)were isolated from four kinds of Lentinus edodes.These glucans were treated with acetone to remove the protein in orderto obtain free protein glucans coded as LNP-I_1,LNP-I_2.LNP-I_3 and LNP-I_4(LNP-I).The free-protein polysaccharideswere sulfated to give derivatives(S-LNP-I)with degree of substitution(DS)from 0.4-0.8.The structural features andweight-average molecular weight(M_w)of the samples were investigated by using infrared spectroscopy,elemental analysis,^(13)C-NMR,size exclusion chromatography combined with laser light scattering(SEC-LLS)and viscometry.The effects ofstructure and conformation of the polysaccharides on antitumor activities were assayed in vivo(Sarcoma 180 solid tumors)and in vitro(Sarcoma 180,HL-60,MCF-7 and Vero tumors).The results indicated that the predominant species of thesamples L-I and LNP-I in 0.2 mol/L NaCl aqueous solution existed as triple-helical chains with high rigidity and in dimethylsulfoxide(DMSO)as single-flexible chains.Interestingly,the antitumor activities of LNP-I are lower than those of the nativeglucans(L-I),whereas their sulfated derivatives have higher inhibition ratio against Sarcoma 180 than LNP-I.The resultsreveal that the binding of protein,sulfated modification and the triple helix conformation are important factors in theenhancement of the antitumor activities of polysaccharides on the whole.

CRITICAL CONCENTRATIONS OF α(1→3)-D-GLUCAN FROM LENTINUS EDODES IN NaOH AQUEOUS SOLUTION

Critical concentrations of α-(1→3)-D-glucan L-FV-Ⅱ from Lentinus edodes were studied by viscometry andfluorescence probe techniques. The dependence of the reduced viscosity on concentration of the glucan in 0.5 mol/L NaOHaqueous solutions with or without urea showed two turning points corresponding to the dynamic contact concentration c_s andthe overlap concentration c~* of the polymer. The values of c_s and c~* were found to be 1×10^(-3) g cm^(-3) and 1.1×10^(-2) g cm^(-3),respectively, for L-FV-Ⅱ in 0.5 mol/L NaOH aqueous solutions. The two critical concentrations of L-FV-Ⅱ in 0.5 mol/LNaOH aqueous solutions were also found to be 1.2×10^(-3) g cm^(-3) fbr c_s and 9.2×10^(-3) g cm^(-3) for c~* from the concentrationdependence of phenanthrene fluorescence intensities. The overlap concentration c~* of L-FV-Ⅱ in 0.5 mol/L NaOH aqueoussolutions was lower than that of polystyrene with same molecular weight in benzene, owing to the fact that polysaccharidetends to undergo aggregation caused by intermolecular hydrogen bonding. A normal viscosity behavior of L-FV-Ⅱ in 0.5 mol/L urea/0.5 mol/L NaOH aqueous solutions can still be observed in an extremely low concentration range at 25℃.

阿拉伯木聚糖、β-D-葡聚糖和α-纤维素涂层对杏果活性氧代谢的影响

使用阿拉伯木聚糖(arabinoxylan,AX)、(1,3;1,4)-β-D-葡聚糖((1,3;1,4)-β-D-glucan,BG)和α-纤维素复合溶液对采后杏果进行涂层处理,探究谷物细胞壁多糖涂层对采后杏果活性氧代谢系统的影响,并阐明其作用机理。结果表明:涂层能够有效抑制丙二醛和活性氧含量的上升,对照组的丙二醛、H2O2和O2-含量分别比ABF-C8处理组(α-纤维素质量浓度8 g/100 mL)高37.50%、33.33%和57.14%;此外,涂层处理还能维持杏果的抗氧化能力和抗氧化酶活性,其抗坏血酸和总酚含量分别比对照组高73.33%和8.40%;小麦细胞壁多糖涂层能够延缓杏果实呼吸过程中活性氧的产生和积累,从而保持细胞膜的完整性,同时涂层能够使水果和外界环境形成气体和水分之间的屏障,维持活性氧清除酶活性及抗氧化能力,从而延长采后杏果的贮藏期。

全自动(1,3)-β-D-葡聚糖检测对老年侵袭性真菌感染的诊断价值

探讨(1,3)-β-D-葡聚糖检测(G试验)自动化检测在老年侵袭性真菌感染(IFI)诊断中的临床意义及价值。方法 采用IGL-200全自动真菌细菌动态检测仪检测440例疑似IFI老年患者血清样本中(1,3)-β-D-葡聚糖含量。结果 440例临床血清样本中阳性59例(阳性率13.41%),阴性381例(阴性率86.59%)。结论 在结合临床表现及病原学检测的基础上,G试验是一种实用的IFI早期诊断指标,能够起到良好的筛查作用,亦能作为老年IFI的动态监测指标,具有较好的临床应用价值。使用全自动设备可以明显提高操作效率和实验准确性,对检验结果更具有保证。

2种中草药静脉注射剂细菌内毒素检查方法的研究

目的 :研究 2种中草药静脉注射剂的细菌内毒素检查方法。方法 :通过试验证明黄芪注射液、刺五加注射液对BET检查均有增强作用。结果 :通过加入过量的 ( 1 3) β D glucan溶液可消除对BET检查的干扰。结论 :此 2种中草药静脉注射剂可以用BET法取代热原检查法。

(1→3)-α-D-葡聚糖的季铵盐合成及其抗菌活性

来源于Penicillium chrysongenum的碱溶性(1→3)-α-D-葡聚糖与缩水甘油基三甲基季铵盐酸盐(glycidyltri methylammonium chloride,GTMAC)反应,生成葡聚糖季铵盐。IR图谱中季铵基团上的甲基的变角振动峰1480cm-1、伸缩振动峰2872cm-1变化明显,1H-NMR表征确定季铵基团在糖环2、4位连接。该葡聚糖的最低抑菌浓度对大肠杆菌为1.0 mg/mL,对金黄色葡萄球菌为2.0 mg/mL。细胞质释放检测结果显示,葡聚糖季铵盐通过作用于细菌细胞膜而发挥抗菌作用。

新型水溶性(1→3)(1→6)-α-D-葡聚糖的性质

利用明串珠菌SK24.002发酵制备水溶性（1→3）（1→6）-α-D葡聚糖,采用高效液相色谱仪、扫描电镜、Zeta电位/粒径仪、热分析系统、差示扫描量热仪、旋转流变仪等现代分析方法测定其理化性质。结果表明,该葡聚糖表面呈多孔网络结构,能溶于水,溶液中粒径分布在40～160nm范围内。相对分子质量分布呈单一峰,具有较高的热稳定性,200℃以下只失去了吸附水,当温度在265～345℃时,发生强烈的热裂解反应,（1→3）（1→6）-α-D葡聚糖溶液的黏度在5～10 g/d L范围内随着质量浓度增加呈指数上升,高浓度的葡聚糖溶液呈现剪切变稀特性,能形成弱凝胶。

水溶性(1→3)(1→6)-α-D-葡聚糖的酶法合成与结构解析

以蔗糖为底物,利用葡聚糖蔗糖酶的聚合反应来制备(1→3)(1→6)-α-D-葡聚糖,通过探讨优化酶促催化条件参数来合成新型葡聚糖,并采用现代分析仪器解析其结构特征。结果表明,葡聚糖蔗糖酶催化反应条件为:反应温度40℃,蔗糖质量浓度160 mg/m L,加酶量10 U/m L,酶反应时间1 h,反应p H 5.0。分子量分布曲线显示葡聚糖分子质量分布集中并呈单峰,绝对重均分子质量2.4×107g/mol,分散系数为1.07,红外与核磁图谱显示糖链中主要由α-1,3和α-1,6连接的D-吡喃葡萄糖单元组成,且两者的比例约为3∶4。

Chronic disseminated candidiasis complicated with a ruptured intracranial fungal aneurysm in ALL

An 11-year-old boy with acute lymphocytic leukemia(ALL) contracted disseminated candidiasis during induction therapy, which was complicated with rupture of a fungal cranial aneurysm. Ventricular drainage and coil embolization of a residual aneurysm in combination with intensive antifungal therapy rescued the patient. Although clinical improvement was achieved, high fever and elevated levels of C-reactive protein and β-D-glucan continued for more than 10 mo. One year later, the ALL relapsed during maintenance therapy with methotrexate and 6-mercaptopurine. After salvage chemotherapy, the patient received unrelated bone marrow transplantation(BMT) in a non-complete remission condition and survived. During subsequent chemotherapy and BMT, no recurrence of the fungal infection was observed under the prophylactic anti-fungal therapy with micafungin.

In situ fluorescence imaging of fungi via(1,3)-β-D-glucan aptamer and tyramide signal amplification technology

Fungal infections are hazardous to human health that has drawn wide attention.In this work,a specific and sensitive method combing the recognition of aptamer to(1,3)-β-D-glucan and tyramide signal amplification technology was proposed for the in situ fluorescence imaging of fungi.Fungi could be distinctly observed by fluorescence microscope rapidly.This method provides morphology and diagnostic information for identifying fungi.The combination of aptamer and tyramide signal amplification technology is a promising tool for the detection of fungi,bacteria and even eukaryotic cell with the virtue of biomarkers.

Effect of cinnamaldehyde on 1,3-β-D-glucans in cell wall of Candida albicans in immunosuppressed BALB/c mice

Objective： Invasive pulmonary candidiasis is a disease with high incidence, difficult treatment, poor prognosis, and high mortality. The present study analyzed the influence of cinnamaldehyde on 1,3-β-D-glucans in the cell wall of Candida albicans in order to provide a theoretical basis for the research of antifungal drugs.Methods： An immunosuppressed BALB/c mouse model with invasive pulmonary candidiasis was established by nasal perfusion of 50 μL of C. albicans suspension（107 cfu/mL）. 1,3-β-D-glucans examination and electron microscopy were carried out. Fluconazole was used as the control.Results： Cinnamaldehyde was administered at a dose of 240 mg/kg/d for 14 consecutive days, and the measured value of 1,3-β-D-glucans was（1160.62 ± 89.65） pg/mL, whereas that of fluconazole was（4285.87 ± 215.62） pg/m L. The difference between the two groups was statistically significant（P 〈 0.05）.Electron microscopy observation indicated that the 2-3 layers outside the cell wall of C. albicans（1,3-β-D-glucans layer） were rough, deformed, and incomplete, although the cell membrane was clear and intact.Conclusion： Cinnamaldehyde demonstrated special efficacy on 1,3-β-D-glucans in the cell wall of C. albicans.

Stereoselective synthesis of the 3,6-branched Fuziα-glucans up to 15-mer via a one-pot and convergent glycosylation strategy

A family of the 3,6-branched Fuziα-glucans including the pentasaccharide repeating unit as well as its di-and trimers were efficiently achieved via a one-pot and convergent glycosylation strategy.All the protectedα-glucans up to 15-mer were assembled with high yields and excellentα-stereoselectivity,which was secured by the synergisticα-directing effects of the Tol SCl/Ag OTf promotion system and the stericβ-facial shielding of bulky saccharide residues linked at the 6-O-position of glucosyl donors.Moreover,the 3,6-branched architecture of glycosyl donor was revealed to be more favorable for theα-selective glucosidation of primary hydroxyl group,especially in the case of large oligosaccharide acceptor.The structurally well-defined syntheticα-glucans would be useful for various biological studies.

In vivo investigation of antimicrobial effects of cinnamaldehyde using immunosuppressed ICR mice with invasive pulmonary aspergillosis

Objective:Invasive pulmonary Aspergillus infection has the characteristics of high morbidity,difficult to be treated,poor prognosis and high mortality.This study aims to investigate the effects of cinnamaldehyde on 1,3-β-D-glucans in the pulmonary Aspergillus fumigatus cell wall to provide a basis for developing novel antifungal drugs.Methods:Immunosuppressed ICR mice were intranasally inoculated with 50μL of A.fumigatus suspension(1×10~7 CFU/m L)and then separated into two groups,for the experimental group cinnamaldehyde was orally administered at 240 mg/kg/d consecutively for 14 d.While for the control group,voriconazole was used to treat the fungus infection.Pulmonary tissues were then extracted for 1,3-β-D-glucans assay and electron microscopy.Results:The concentration of 1,3-β-D-glucans was significantly different between the cinnamaldehyde and voriconazole groups,which was(1160.89±364.96)pg/m L and(3885.94±845.45)pg/m L,respectively(P<0.01).Electron microscopy showed that 2-3 outer layers(1,3-β-D-glucan layer)of A.fumigatus cell wall were damaged and fell off,resulting in serious defect of the cell wall,but the cell membrane was clear and intact.Conclusion:Cinnamaldehyde has a significant influence on the integrity of 1,3-β-D-glucans in the pulmonary A.fumigatus cell wall,but the cell membrane is unaffected,suggesting that cinnamaldehyde has unique antifungal properties depending on its action against the 1,3-β-D-glucans on the pulmonary A.fumigatus cell wall.